Hepatitis C virus (HCV) is a leading cause of liver disease and liver cancer. Nearly 170 million worldwide are chronically infected with HCV, and current treatment options are effective for only half of the infected. HCV persistence is mediated by the ability of the virus to evade the host innate immune response. While HCV RNA is sensed as foreign by the host cell cytosolic receptor RIG-I, the downstream signaling induced by this detection is suppressed by HCV NS3/4A protease cleavage of MAVS, the RIG-I signaling adaptor. MAVS is localized to mitochondria and peroxisomes, and I have recently found that it is also localized to mitochondrial- associated ER membranes (MAM), which link mitochondria and peroxisomes together into synapses during RIG-I activation. Further, I have found that the HCV NS3/4A protease targets these sites via the NS4A subunit of the protease complex for cleavage of MAVS on the MAM, and not the mitochondria. This data strongly implicates MAVS with MAM localization as the signaling active molecule that drives the innate immune response towards HCV. This proposal will explore the hypothesis that molecules targeted to the MAM during RIG-I pathway activation induce the formation of a MAVS signaling complex by using biochemical and proteomic studies that identify MAM-recruited molecules during the host innate immune response. In addition, the spatial-temporal regulation of NS4A targeting of the NS3/4A protease complex to the MAM for MAVS cleavage and immune evasion will be determined by identifying both the host cell factors and viral components that guide this targeting. These studies will provide valuable insights into the mechanism that activates the switch between the role of NS3/4A in HCV replication and innate immune control. Moreover, they will form the basis for studies to understand how MAM-targeting of MAVS by NS3/4A supports persistent HCV infection. These studies will determine how the MAM organizes both innate immune signaling and the HCV innate immune evasion program. A detailed understanding of these processes will have implications for therapeutic and vaccine design strategies to limit infection by HCV and other RNA viruses that are sensed by the RIG-I pathway.